| Chronic endemic arsenism via drinking water, is a chronicity cumulative toxicosis disease and becomes an international health hazard. In order to reduce the adverse health effects of arsenism, the central and local governments of China have provided significant funds to change water levels of As and at the same time take general measures to"reduce arsenic intake, remove arsenic from the body and treat the patients". However, the pathogenesis of arsenism is not clear and the research progress on the sensitive responsive biomarkers for arsenic exposure is not satisfactory either. To elucidate the molecular mechanisms through which the arsenic causes injury of blood vessel, we designed a series experiments to detect the effect of Sodium Arsenite of low doses on the apoptosis, the relationship between oxidative stress or Mitochondria membrane potential and apoptosis respectively and the possibly involved mechanisms, using HUV-EC as the model system. We screen the expression of early responsive genes of the arsenicosis, and find several sensitive biomarkers for arsenic exposure.Methods:After treated with different concentrations of sodium arsenite, Mitochondrial membrane potential alteration of HUV-EC is assessed by the retention of rhodamine 123 (RH123), a specific fluorescent cationic dye that is readily sequestered by active mitochondria, depending on their transmembrane potential. Also the ROS content is detected by the DCFH-DA staining and the cell apoptosis is measured using Annexin V-PI kit. Real time-PCR analysis is performed to screen the possible target genes which are responsive when HUV-ECs are treated with sodium arsenite of different low doses. Results:One: confocal laser microscope images show that cells modulate from polygonal shape to the round shape as the concentration of sodium arsenite increases while the cell adhesiveness decreases, and the cell debris without clear outlines is observed .Two: the sodium arsenite causes the mitochondrial membrane potential alteration, and the alteration decreases when the dose of sodium arsenite increases, with a obvious alteration for MMP at 5μM sodium arsenite.Three: Flow cytometry analysis shows that the apoptosis pattern is dose-dependent in which the apoptosis increases as the concentration of sodium arsenite increases.Four: the ROS expression pattern is double dose-dependent, when HUVECtreated with different concentrations of sodium arsenite.Five: two responsive genes, SOD2 and NQO1, are identified by real time-PCR analysis that the expressions of the two genes go up when HUVECtreated with sodium arsenite of low concentrations.Conclusions:One: sodium arsenite is able to change the appearance of HUVECand induce apoptosis.Two: the ROS level is double dose-dependent, with a typical S responsive curve, suggesting that antioxidant defense system is active when of HUVEC treated with sodium arsenite.Three: The ROS level is affected before MMP when HUVEC treated with sodium arsenite, suggesting that the ROS is more sensitive than the MMPFour: The minimal dose of sodium arsenite needed to induce the apoptosis is the same needed to induce the MMP alteration, suggesting that the MMP alteration might be a major pathway of cell apoptosis.Five: The expressions of SOD2 and NQO1 increase strikingly when HUVEC treated with a low concentration of sodium arsenite (5μM) and this fact might indicate a basis for our extended research on the sensitive responsive biomarkers by arsenic exposure.Collectively, the apoptosis caused by arsenic and the ROS level alteration are very important during the early injury of blood vessel by arsenism. The fact that, the expressions of SOD2 and NQO1 increase strikingly and the ROS level decreases when HUVEC treated with a low concentration of sodium arsenite, suggests that SOD2 and NQO1 are very functionally important in down regulation of the ROS level and thus protection of the blood vessel from the injury caused by the arsenism.
|
PDF Full Text Request